Stage-specific requirement for METTL3-dependent m6A modification during dental pulp stem cell differentiation

Background N6-methyladenosine (m6A) is the most prevalent epigenetic modification in eukaryotic messenger RNAs and plays a critical role in cell fate transition. However, it remains to be elucidated how m6A marks functionally impact the transcriptional cascades that orchestrate stem cell differentiation. The present study focuses on the biological function and mechanism of m6A methylation in dental pulp stem cell (DPSC) differentiation. Methods m6A RNA immunoprecipitation sequencing was utilized to assess the m6A-mRNA landscape during DPSC differentiation. Ectopic transplantation of DPSCs in immunodeficient mice was conducted to verify the in vitro findings. RNA sequencing and m6A RNA immunoprecipitation sequencing were combined to identify the candidate targets. RNA immunoprecipitation and RNA/protein stability of Noggin (NOG) were evaluated. The alteration in poly(A) tail was measured by 3′-RACE and poly(A) tail length assays. Results We characterized a dynamic m6A-mRNA landscape during DPSC mineralization with increasing enrichment in the 3′ untranslated region (UTR). Methyltransferase-like 3 (METTL3) was identified as the key m6A player, and METTL3 knockdown disrupted functional DPSC differentiation. Moreover, METTL3 overexpression enhanced DPSC mineralization. Increasing m6A deposition in the 3′ UTR restricted NOG expression, which is required for DPSC mineralization. This stage-specific m6A methylation and destabilization of NOG was suppressed by METTL3 knockdown only in differentiated DPSCs. Furthermore, METTL3 promotes the degradation of m6A-tagged NOG by shortening the poly(A) tail length in the differentiated stage. Conclusions Our results address an essential role of dynamic m6A signaling in the temporal control of DPSC differentiation and provide new insight into epitranscriptomic mechanisms in stem cell-based therapy. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-022-03814-9.


Introduction
Mesenchymal stem cells (MSCs) residing in various tissues can still undergo self-renewal and differentiate into specific cell types to maintain tissue homeostasis and fulfill regenerative needs [1]. Multiple adult stem cell types with diverse biological properties have been reported in many tissues and organs. Beyond their similarity in cellular and molecular functions, MSCs exhibit distinct features related to their original phenotypes. Dental pulp stem cells (DPSCs) have superior pluripotency capacity and high mineralization potential, which are essential for hard tissue formation [2]. DPSCs can differentiate into odontoblasts and secrete mineralized matrix known as tertiary dentin bridge formation to seal the vital pulp chamber and prevent pulpal infection from potential insult [3]. The differentiation of DPSCs is critical for *Correspondence: shaolongquan@smu.edu.cn Page 2 of 15 Luo et al. Journal of Translational Medicine (2022) 20:605 tertiary dentin formation and dental repair in vital pulp therapy [3,4]. Meanwhile, accumulating evidence also supports that DPSCs are capable of differentiating into osteoblasts and forming lamellar bone, which is a promising stem cell source in bone engineering [5]. The initiate signaling reprograms cellular differentiation and extracellular matrix secretion will greatly benefit therapeutic approaches in engineering DPSC-based vital pulp procedures. Epigenetic modulations are capable of temporally controlling the transcription programs in a heritable manner and subsequently guiding quiescent stem cells to undergo preferential trajectories toward differentiation [6]. The key epigenetic regulators could reversibly modulate endogenous stem cell activities and promote functional mineralized tissue formation, which present therapeutic opportunities in regenerative strategies. N 6 -methyladenosine (m 6 A) is the most prevalent posttranscriptional modification of messenger RNA (mRNA) and regulates almost every step of RNA metabolism in mammals [7]. The methyltransferase complex formed by methyltransferase-like 3 (METTL3), METTL14, and Wilms tumor 1-associated protein (WTAP) is responsible for the m 6 A modification of mRNA, which catalyzes adenosines with methyl groups from metabolite substrates [8]. Large-scale transcripts are dynamically and timely tagged by m 6 A marks to orchestrate the different stages of stem cells. RNA m 6 A modification has emerged as a critical epitranscriptomic mechanism that regulates embryonic development, cell reprogramming and differentiation [9,10]. The disruption of m 6 A modification in the stem cell program displays diverse effects across cell types and specific fate stages. Reduction of m 6 A deposition improved pluripotency and blocked regeneration of embryonic stem cells [11], while it limited self-renewal and triggered cell differentiation in epiblast stem cells and induced pluripotent stem cells [12,13]. It is critical to identify the regulatory mechanism of dynamic m 6 A marks in the DPSC fate transition. Our previous study characterized the m 6 A-tagged landscape in immature DPSCs, which is related to cell senescence and apoptosis [14]. Meanwhile, how m 6 A methylation participates in DPSC differentiation remains unclear. Clarifying the RNA epigenetic mechanism during DPSC differentiation and manipulating the key modulators in therapeutic applications would advance vital pulp therapy.
In this study, we revealed a dynamic and unique m 6 A-mRNA landscape with m 6 A RNA immunoprecipitation-sequencing (m 6 A RIP-seq), which provides an entry point to uncover the potential function of m 6 A methylation in DPSC differentiation. METTL3 was identified as a key molecule that mediates m 6 A modification in DPSC mineralization. Remarkably, dynamic m 6 A methylation of noggin (NOG) confers its stabilization by shortening the poly(A) tail in a stage-specific manner.
Our results provide evidence for the critical role of m 6 A modification in DPSC differentiation and shed light on the epitranscriptomic mechanism in the temporal control of cell fate transition.

DPSC isolation and culture
Dental pulp tissues were collected according to the appropriate guidelines after written informed consent was obtained by a protocol approved by the Human Research Committee of Stomatological Hospital, Southern Medical University (ethical code 2019 (16)). Primary DPSCs were harvested and cultured as previously described [15]. Briefly, dental pulp tissues were removed from extracted third molars and then digested with 3 mg/ mL collagenase I (Gibco-Invitrogen, Carlsbad, CA, USA). DPSCs were collected and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (all from Gibco-Invitrogen) at 37 °C with 5% CO 2 . The medium was changed every 2 days, and DPSCs at passages 3-5 were used for subsequent experiments. DPSCs (at a density of 2 × 10 5 cells/mL) were seeded in 6-well or 24-well plates (Corning Life Sciences, NY, US) and cultured until the cells reached 80-90% confluence. The culture medium was changed to osteo/ odontogenic medium (OM) containing 10 mmol/L β-glycerophosphate, 50 μg/mL ascorbic acid and 0.1 μmol/L dexamethasone (all from Sigma-Aldrich, St. Louis, MO, USA). The induction medium was changed every other day from day 0 to day 21 (the day of induction was defined as day 0). After 7 days of induction, the cells were fixed and stained for alkaline phosphatase (ALP) (Beyotime Biotechnology, Shanghai, China) (n = 5). Alizarin red staining (ARS) (Beyotime) was used to stain the accumulated mineralized matrix on day 14 (n = 5). For adipogenic differentiation, DPSCs were induced by the Adipogenesis Differentiation Kit (HUXXC-90031, Cyagen Biosciences, Guangzhou, China). Cells were cultured with adipogenic inducing solution A for 3 days, and the medium was replaced with solution B for 1 day and then replaced with solution A. This cycle was repeated four times and then subjected to oil red O staining (Cyagen Biosciences) (n = 5).

Total m 6 A and methylated metabolite measurements
DPSCs were subjected to osteo/odontogenic induction for 0, 7, and 14 days, and RNA was extracted using TRIzol solution (Takara Biotechnology, Shiga, Japan). Total m 6 A was measured with an m 6 A RNA methylation quantification kit (P-9019-96, Epigentek, Farmingdale, NY, US) according to the manufacturer's protocol. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was utilized to assess the metabolite compounds related to m 6 A methylation, including S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). Cell samples were subjected to methanol and homogenized before analysis with an ultrahigh-performance liquid chromatography (UHPLC) column (1290 Infinity LC, Agilent Technologies). Next, during MS/MS acquisition, a m/z range of 25-1000 Da was used, and the ion accumulation time was screened. Metabolite compounds were identified with a database of available standards and subjected to multivariate data analysis. Metabolites with a variable importance in projection (VIP) value > 1 were further subjected to statistical analysis. m 6 A RIP-seq and m 6 A RIP-qPCR m 6 A RIP-seq was used to characterize m 6 A modification during DPSC mineralization (osteo/odontogenic medium-induced DPSCs, OM-DPSCs). Total RNA was isolated from DPSCs induced for 0, 7, and 14 days with TRIzol solution. The extracted RNA enriched and purified with oligo(dT)-attached magnetic beads and an m 6 A RIP kit (17-10499, Millipore, Burlington, MA, US) according to the kit's protocol. The purified m 6 A-RIP RNA fragments were then fragmented into small pieces with fragmentation buffer for sequencing. The RNA fragments were incubated with magnetic beads conjugated with an m 6 A-specific antibody in buffer. The IP RNA and input RNA were reverse transcribed into cDNA and subjected to deep sequencing on an Illumina NovaSeq ™ 6000 platform for m 6 A RIP-seq [16]. m 6 A peak calling, distribution, motif mapping and enrichment analysis were performed by LC-BIO Technologies Co., Ltd. (Hangzhou, China). To identify specific genes targeted by METTL3, the enrichment of m 6 A-modified Noggin (NOG) mRNA in the immunoprecipitate (IP) and input RNA was quantified by qPCR analysis as described previously for m 6 A RIP-qPCR [14].

RNA sequencing and profile analysis
Total RNA was extracted from DPSCs, and poly(A) mRNA was purified with poly(T)-conjugated magnetic beads. Then, the mRNA was fragmented into small pieces and converted into double-stranded cDNA. Paired-end runs with a read length of approximately 300 base pairs (bp) were used for RNA-sequencing with the Illumina deep sequence platform by LC-BIO Technologies Co., Ltd. Significantly differentially expressed genes (DEGs) were identified in this study as genes with a fold change in expression ≥ 2.0 and corresponding p value < 0.05. The DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses with the R packages GOseq and DAVID.

Quantitative polymerase chain reaction (qPCR)
The cells were digested and total RNA was extracted from DPSCs with a Total RNA Isolation Kit (Foregene Biotechnology, Chengdu, China). The RNA was then reverse transcribed with RT Master Mix (Takara) according to the manufacturer's instructions to obtain complementary DNA (cDNA). Real-time qPCR was performed with TB Green qPCR Mix (Takara) according to the manufacturer's protocol. Relative target gene expression was analyzed with a standard curve and normalized to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) expression. The primer sequences used in qPCR are summarized in Additional file 1: Table S1.

Animal model construction
The animal experiments were conducted in compliance with ARRIVE guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures followed protocols approved by the Ethics Committees of Stomatological Hospital, Southern Medical University (ethical code 2019 (16)). For ectopic transplantation studies, porous beta-tricalcium phosphate/hydroxyapatite (β-TCP/HA) discs (diameter: 4 mm, thickness: 2 mm) were obtained from Biological Materials Manufacturing Core, Sichuan University. Approximately 1 × 10 6 transfected DPSCs were seeded on β-TCP/HA discs and cultured in a 24-well plate with odontogenic medium for 24 h [17,18]. The composites of DPSCs and the β-TCP/HA scaffold were transplanted into the subcutaneous dorsal pockets of 6-week-old BALB/c immunodeficient nude mice (n = 10) [18,19]. Two subcutaneous pockets were made on the right and left side of the dorsal surface, each allowing for one composite. The shCTR and shMETTL3 groups were carefully transplanted into the left and right subcutaneous regions, respectively (n = 5), as were the LV-METTL3 and LV-CTR groups (n = 5). After 4 weeks, the harvested implants were fixed with paraformaldehyde, followed by decalcification with Ethylenediaminetetraacetic acid (EDTA) for 2 weeks. The formation of new mineralized tissue was evaluated by Masson-trichrome staining.

m 6 A site-specific mutant plasmid construction
To investigate the biological effect of the m 6 A methylated sites in the 3′ untranslated region (UTR) of NOG, full-length NOG cDNA was constructed and cloned into the NheI-and BamHI-digested Flag-PCDNA3.1(+) vector by Genecreate Biotechnology Co., Ltd. (Wuhan, China). The vector used to express NOG with the wildtype m 6 A motif was referred to as NOG-WT. Two different mutants (NOG-MUT1 and NOG-MUT2) were generated by introducing single-nucleotide mutations (A to T) in the four m 6 A motifs of the 3′ UTR after mapping the conserved m 6 A motif sequence in m 6 A RIP-seq. To examine mRNA expression, 293T cells were transfected with NOG-WT vector or a mutant NOG vector and subjected to qPCR [20].

RNA and protein stability analysis
RNA transcription in DPSCs was inhibited by treatment with 5 µg/mL actinomycin D (ActD, 7240-37-1, Sigma-Aldrich) as described in a previous study to analyze mRNA decay rates [21]. mRNA was isolated after 0, 4, and 8 h and subjected to qPCR. The half-life of NOG mRNA has been reported.
Protein translation in DPSCs was inhibited by treatment with 100 µg/mL cycloheximide (CHX, A8244, APExBIO Technology, Houston, TX, US) to analyze protein stability. Total protein was isolated after 0, 4, and 8 h and subjected to western blot analysis. The protein expression level of NOG was used to analyze its stability.

3′-Rapid amplification of cDNA ends (3′-RACE)
3′-RACE to obtain the 3′ UTR sequences of NOG from shMETTL3 or shCTR DPSCs after osteogenic induction was performed with a 3′-RACE kit (6106, Takara) according to the protocol. The 3′-RACE products after two rounds of amplification were purified and subcloned into a vector, and the amplified fragments were further identified by sequencing [22]. The NOG-specific primers

Poly(A) tail assay
A poly(A) tail assay was performed with a Poly(A) Tail-Length Assay Kit (764551KT, Thermo Fisher) according to the protocol. Briefly, poly(A) polymerase was used to add G and I to the 3′ ends of the RNA, and the newly tailed RNA was converted to cDNA by reverse transcription. Then, NOG-specific forward and reverse primers and universal reverse primers were used to generate a product consisting of NOG with a poly(A) tail. The PCR products from METTL3 knockdown DPSCs in differentiated/undifferentiated stages were separated on agarose gels [23]. The specific primers for NOG were F: 5′-TAA CCT GCT ATT TAT ATT CCA GTG CCC TTC -3′ and R: 5′-TGA ACT CTA TAG CTT CTT CGA GGT CCAA-3′.

Quantification and statistical analysis
The experiments in this study were carried out biologically repeated at least three times, and the data are presented as the mean ± standard deviation. Statistical differences were evaluated by one-way analysis of variance (ANOVA) and corresponding post hoc tests for multiple comparisons. Unpaired two-tailed Student's t test was used to compare two groups. A p value < 0.05 was considered to be statistically significant and was analyzed by GraphPad Prism 7.0 (La Jolla, CA, US).

The dynamic m 6 A epitranscriptomic landscape in DPSC differentiation
We first examined the total m 6 A content and epitranscriptomic profile of m 6 A-tagged mRNA. The total m 6 A level in the RNA pool was upregulated upon osteo/ odontogenic induction for 7 and 14 days, as quantified by a colorimetric method (Fig. 1A). m 6 A RIP-seq analysis showed that the majority of m 6 A peaks were enriched in the DNA coding sequence (CDS), 3′ UTR and stop codon, with small subsets located in the 5′ UTR and start codon (Fig. 1B, C). Notably, the density of m 6 A peaks in the 3′ UTR increased over time, and the representative motif sequence of m 6 A sites also underwent slight modification (Fig. 1B-D). Next, conjoint analysis of m 6 A RIP and RNA-seq was used to identify the differentially m 6 A-methylated and expressed molecules and some representative genes were marked (Fig. 1E). These m 6 A-tagged genes were found to be mainly enriched in biological processes related to signal transduction, transcriptional regulation and cell differentiation (Fig. 1F). The signaling pathways as transforming growth factor-β (TGF-β) and Rap1 were identified in KEGG pathway analysis (Additional file 1: Fig. S1A). The establishment of m 6 A marks relies on the riboswitch from the methyl donor SAM to SAH conducted by the methyltransferase complex. We next evaluated the methylation potential and expression pattern of m 6 A-related molecules during DPSC mineralization. The concentrations of the methyl metabolite (SAH) were increased after induction, while the methyl donor (SAM) and methylated potential (SAM/SAH ratio) showed no significant variation (Fig. 1G). RNA-seq analysis showed that the relative gene expression of METTL3 was markedly increased on days 7 and 14 of induction among all the m 6 A-related molecules (Additional file 1: Fig.  S1B). Regarding methyltransferases, the mRNA and protein levels of METTL3 also increased after odontogenic induction and peaked on day 14, while METTL14 exerted low protein expression levels, and WTAP showed no obvious trend (Fig. 1H, I). Taken together, these data suggest that METTL3 is the key player in m 6 A modification during DPSC mineralization.

METTL3 is required for functional DPSC differentiation
To identify the potential impact of METTL3 deletion on DPSC differentiation, two shRNA lentiviral vectors were used to knockdown METTL3 expression and both sufficiently suppressed the mRNA and protein expression by more than half ( Fig. 2A, B). ALP, RUNX2 and DSPP were also downregulated at the mRNA level (Fig. 2C). The protein expression of RUNX2 was significantly reduced after 3 and 14 days of induction, and DSPP was also suppressed on days 3 and 7 (Fig. 2D). METTL3 knockdown led to weaker ALP activity and less mineralized nodule formation after 7 and 14 days of odontogenic induction (Fig. 2G, H). These results indicated that METTL3 inhibition impaired the odontogenic differentiation of DPSCs. Meanwhile, the expression of METTL3 was suppressed during adipogenic induction (Fig. 2E). METTL3 inhibition enhanced the mRNA expression of Lipoprotein Lipase (LPL) and Peroxisome Proliferator Activated Receptor (PPAR) and promoted lipid droplet formation (Fig. 2F, I). Overall, METTL3 inhibition compromised odontogenesis but promoted adipogenesis, implying a critical role of m 6 A methylation and METTL3 in controlling DPSC differentiation.

METTL3 overexpression enhances the mineralization of DPSCs
Considering the negative effect of METTL3 inhibition, we hypothesized that METTL3 overexpression would benefit DPSC mineralization. The elevated mRNA and protein levels of METTL3 upon overexpression lentivirus transfection were confirmed (Fig. 3A, B). METTL3 overexpression induced ALP activity and calcium nodule formation (Fig. 3C). The favorable effect of METTL3 overexpression was also evidenced by the increased mRNA expression of ALP, RUNX2, and DSPP and protein expression of RUNX2 and DSPP (Fig. 3D, E). These results support the promoting effect of METTL3 overexpression on DPSC odontogenic differentiation and matrix mineralization.
To evaluate the role of METTL3 in DPSC mineralization in vivo, we conducted subcutaneous transplantation of β-TCP/HA scaffolds with DPSCs in BALBc nude mice. DPSCs were subjected to METTL3 knockdown or overexpression prior to the incubation with β-TCP/HA scaffolds. Hematoxylin and eosin staining was performed to evaluate cellularity alteration (Additional file 1: Fig.  S2) and Masson-trichrome staining assess extracellular matrix production. Immature mineralized tissue and collagen fiber (blue staining) formation were reduced by approximately two-thirds in the METTL3 knockdown DPSC groups compared to the control groups (Fig. 3F,  H). Moreover, METTL3 overexpression enhanced collagen tissue formation by more than twofold after 4 weeks (Fig. 3G, H), indicating the therapeutic potential of METTL3 in DPSC differentiation and mineralized tissue formation.

Dynamic m 6 A modification of NOG orchestrates the differentiation stage
To dissect the potential targets of METTL3-mediated m 6 A modification in DPSC differentiation, transcriptome sequencing was used to screen the gene expression profile after METTL3 deletion in OM-DPSCs (Additional file 1: Fig. S3). GO analysis showed that these DEGs were mainly related to mRNA 3′ UTR binding, signal transduction and cell differentiation (Fig. 4A, B), which corresponds to the elevated m 6 A accumulation in the 3′ UTR during DPSC mineralization. Signaling pathways regulating the pluripotency of stem cells, TGF-β and Wnt signaling pathways were also enriched in the KEGG pathway analysis (Fig. 4C). In the m 6 A-RIP and transcriptome sequencing of DPSC mineralization, 279 genes with significant differences in both m 6 A peaks and expression were identified (Figs. 1E, 4D). Next, we filtered the potential m 6 A target genes of METTL3 by taking the intersection of these 279 genes and the shMETTL3-related DEGs in both the early and late stages of differentiation (Fig. 4E). Among the six candidate targets, NOG is a wellknown inhibitor of bone morphogenetic protein (BMP) signaling and the downstream Smad pathway. The gene and protein expression levels of NOG were significantly reduced by half in DPSCs upon odontogenic induction, which was consistent with the transcriptome sequencing data (Fig. 5A, B).
Immunofluorescence staining showed that the cytoplasmic expression of NOG was significantly suppressed after induction (Additional file 1: Fig. S4A). Moreover, the colocalization coefficient of METTL3 and NOG was reduced in differentiated DPSCs (Additional file 1: Fig. S4B). Visualization of m 6 A RIP-seq data showed that high-confidence and specific m 6 A peaks in the 3′ UTR of NOG mRNA markedly increased after induction (Fig. 5C). The two ascending m 6 A peaks in the 3′ UTR of NOG appeared to be the center of four m 6 A sites after mapping the conserved motif sequence. To explore the potential impact of these m 6 A marks on gene expression, NOG expression vectors containing wild-type m 6 A sites (WT) and two A-to-T m 6 A mutations (MUT1 and MUT2) were constructed (Fig. 5D). Both m 6 A mutations resulted in increased mRNA expression of NOG compared with WT expression in 293 T cells (Fig. 5E). These data demonstrated that m 6 A tag accumulation in the 3′ UTR of NOG resulted in diminished gene expression, which is required for DPSC differentiation. Next, we investigated the potential effect of METTL3 depletion on m 6 A-tagged NOG. The m 6 A methylation level of NOG decreased sharply after METTL3 knockdown in OM-DPSCs (Fig. 5F), indicating that the elevated m 6 A enrichment in NOG was mediated by METTL3. In the ectopic mineralization models, the composites of β-TCP/HA scaffolds with METTL3-knockdown DPSCs showed a remarkable upregulation in NOG expression (Fig. 5G, I), while the NOG secretion were significantly suppressed in METTL3-overexpression transfected composites after subcutaneous transplantation (Fig. 5H, I).

Stage-specific NOG expression is regulated by METTL3
NOG antagonizes BMP signaling and selectively inhibits BMP-related Smad pathway activity, which is essential for cell differentiation. Notably, METTL3 knockdown enhanced the relative expression of NOG in the differentiated state but not in the undifferentiated state, and qPCR confirmed the inhibitory effect of METTL3 on NOG mRNA expression in only OM-DPSCs (Fig. 6A). Additionally, METTL3 deletion significantly upregulated the protein expression of NOG in OM-DPSCs, and METTL3 overexpression exerted the opposite effect (Fig. 6B). Meanwhile, no stable trend was observed for NOG expression changes in undifferentiated DPSCs. These data suggested that METTL3 regulated m 6 A-tagged NOG expression to orchestrate the differentiation process. Furthermore, we evaluated the downstream signal transduction of NOG by assessing phosphorylation of Smad1/3/5 [24]. METTL3 deletion suppressed the phosphorylation of Smad3 and Smad1/5, while METTL3 overexpression enhanced the activation of the Smad pathway in differentiated DPSCs (Fig. 6C). Consistently, the phosphorylation level of Smad3 were dramatically inhibited in METTL3knockdown DPSCs with β-TCP/HA scaffolds, while significantly enhanced in METTL3-overexpression composites in the ectopic mineralization models (Additional file 1: Fig. S5). The impaired ALP activity and calcium mineralization formation induced by METTL3 inhibition were significantly upregulated after neutralizing NOG protein with a blocking antibody (Fig. 6D), as was the mRNA expression of RUNX2 and DSPP (Fig. 6E). These data demonstrated that neutralizing excess NOG secretion can partially rescue insufficient odontogenic differentiation of METTL3-deleted DPSCs.

METTL3 programs NOG destabilization via poly(A) tail shortening
Clustered m 6 A hallmarks in the 3′ UTR around the stop codon mainly influence the stability and localization of RNA. RNA stability assays with actinomycin D to suppress transcription showed that METTL3 knockdown increased the half-life of NOG mRNA in differentiated DPSCs (Fig. 7A). Furthermore, in the undifferentiated state, the mRNA decay of NOG was not significantly decelerated (Fig. 7A). Then, we blocked protein translation and synthesis by cycloheximide. The protein degradation of NOG in differentiated DPSCs was also suppressed after METTL3 inhibition (Fig. 7B). Consistent with previous data, METTL3 specifically restricted the stabilization and promoted the degradation of m 6 A-tagged NOG in differentiated DPSCs. The poly(A) tail determines the mRNA stability and translation initiation of most eukaryotic mRNAs. Transcriptome sequencing suggested the presence of only one transcript of NOG without alternative splicing, which is in line with the transcript reported in the National Center for Biotechnology Information (NCBI) database (reference sequence: NC_000017.11). 3′-RACE analysis was conducted to obtain the 3′ UTR sequence information of NOG after METTL3 depletion. The 3′-RACE results showed no obvious alterations in the polyadenylation site or 3′ UTR length, while the number of A residues appeared to be increased in OM-DPSCs (Fig. 7C, D). Traditional RACE might result in PCR bias in the poly(A) tail due to the use of universal adaptor primers and nested amplification [22]. The poly (A) tail length was further quantified by PCR with NOG-specific primers after random oligomer addition of guanosine and inosine residues. The poly(A) tail was dozen bps longer after METTL3 knockdown in differentiated DPSCs, but this difference was not detected in undifferentiated DPSCs (Fig. 7E). These data support that METTL3 exerts temporal control over NOG stabilization to orchestrate DPSC differentiation (Fig. 7F).

Discussion
Emerging evidence has proven that m 6 A RNA methylation is a critical epitranscriptomic mechanism that permits additional specificity and plasticity to the transcriptome [25]. Here, we revealed the dynamic and unique m 6 A mRNA landscape in DPSC mineralization, and elevated m 6 [8,26]. During embryonic cortical neurogenesis, m 6 A-methylated transcripts are enriched in biological processes, such as neural stem cells, the cell cycle, and differentiation, which are essential to control the transcriptome composition of different stages [25]. We characterized the dynamic and unique m 6 A landscape in DPSC mineralization, and the m 6 A-mRNA profile was mainly related to transcriptional regulation and cell differentiation. Moreover, the increasing total m 6 A content and m 6 A distribution in the 3′ UTR might result from a pronounced elevation of METTL3 expression. METTL3 was reported to participate in tooth root development by modulating translational efficiency [27]. The inhibition of DPSC proliferation and osteogenesis by METTL3 knockdown was associated with an impaired glycolytic pathway [28]. METTL3 is also involved in bone mesenchymal stem cell (BMSC) differentiation and function [16,29]. METTL3 depletion in BMSCs impaired osteogenic differentiation, while METTL3 overexpression partly abrogated the induction of osteoporosis in mice [16]. Consistent with the current literature, METTL3 inhibition comprised DPSC differentiation, and METTL3 overexpression facilitated DPSC mineralization, indicating therapeutic potential. There are several in vivo models available to verify the regulatory mechanism of DPSC differentiation, such as dentin-pulp complex regeneration in situ and ectopic transplantation of DPSCs [30,31]. Ectopic mineralization models were used in this study and subcutaneous transplantation in immunocompromised mice supported that METTL3 is a positive regulator of DPSC differentiation and mineralized tissue formation. More evidences from orthotopic models are needed to support the therapeutic application in vital pulp procedures and DPSC-based therapy. Various transcripts and signals are tagged in a timely manner by m 6 A modification, which in turn controls proper development and differentiation. METTL3-mediated m 6 A modification regulates the expression of some osteogenic markers and other related genes involved in bone metabolism [32]. Parathyroid hormone (PTH)/ Pth1r, TGF-β/SMAD, WNT and other signaling pathways are modulated by m 6 A marks, which are essential in the cellular differentiation and cancer development [16,33,34]. Here, NOG and downstream Smad pathway were identified as the target of METTL3-mediated m 6 A modification during DPSC differentiation. NOG is a key player in ectoderm development, and its disruption can lead to organogenesis defects such as craniofacial defects and hypoplastic teeth [35,36]. Noggin is capable of binding and inactivating members of the TGF-β superfamily proteins as BMPs, subsequently blocking BMP-induced Smad pathway activation [37]. BMSC osteogenesis and DPSC odontogenesis are regulated by NOG via the downstream Smad1/5 signaling pathway [38,39]. We found that the m 6 A peaks in the 3′ UTR of NOG mRNA increased during DPSC mineralization, which restricted its gene expression. METTL3 inhibited m 6 A-tagged NOG expression and promoted its degradation in differentiated DPSCs. Consistent with our data, m 6 A modification modulates RNA degradation and gene expression in neural stem cells, which is a critical epigenetic mechanism in the temporal control of neurogenesis [25]. m 6 A signaling clustered in the 3′ UTR is mainly responsible for cytoplasmic events related to RNA stability and translation [40][41][42], and METTL3 can independently read and modulate m 6 A marks in the 3′ UTR of certain transcripts [40]. Taken together, these findings suggest that m 6 A modification dynamically modulates the stability of specific transcripts, which is required for the transcriptional prepatterning of DPSC mineralization.
Poly(A) tails are 150-250 adenosine nucleotides acquired by the end of the 3′ UTR in the nucleus that subsequently undergo deadenylation in the cytoplasm. The length of a poly(A) tail changes throughout the lifetime of mRNA and has essential effects on its stability, degradation and translation [43]. In the global transcriptome, transcripts with a longer poly(A) tail possess a longer average mRNA half-life [23]. The deadenylation of shorter poly(A) tails can cause RNA decay or translational defects [44]. A recent study also noted the correlation between m 6 A marks and poly(A) tail regulation. The transcriptional dynamics of certain genes are related to differences in poly(A) tail length via m 6 A modification and deadenylase complexes [45]. m 6 A signaling is capable of controlling RNA structural switching and RNA-protein interactions [46]. METTL3 and WATP can modulate RNA stabilization in an m 6 A-HuR-dependent manner [47,48]. The m 6 A reader YTH N 6 -methyladenosine RNA binding protein (YTHDF) 2 is reported to directly interact with the CCR4-NOT complex. YTHDF3 can recruit the poly(A) specific ribonuclease subunit (PAN) 2-PAN3 complex, contributing to its deadenylation and degradation [49,50]. Nonadenosine residues, such as G modifications, are also related to high quality and delayed degradation of the poly(A) tail [51,52]. In our study, the temporal control of NOG stabilization by METTL3 relied on poly(A) tail shortening in the differentiation stage. Further studies are needed to identify the specific mechanism of how m 6 A marks lead to shortened poly(A) tails in DPSC differentiation. The osteo/odontogenic differentiation of DPSCs and tertiary dentin formation are of particular interest in relation to dental repair. Identifying the key signaling in DPSC differentiation and mineralized matrix formation, and recapitulating these processes in clinical strategies could preserve pulp vitality. In the present study, we demonstrated that dynamic m 6 A RNA methylation is essential for heightened transcriptional coordination during DPSC differentiation. METTL3-mediated m 6 A marks tag the 3′ UTR of NOG and inhibit its stabilization via poly(A) tail regulation in a stage-specific manner. The present study identifies a critical role of METTL3mediated m 6 A methylation in the temporal control of cell fate transition and sheds light on the epitranscriptomic machinery of m 6 A-dependent poly(A) tail regulation in transcriptional dynamics.